DESCRIPTION (applicant's abstract) The amyloid precursor proteins (APPs) are a family of type I transmembrane glycoproteins containing a extracytoplasmic regions, a transmembrane sequence, and a small cytoplasmic domain. The proteins play important roles in the development of Alzheimer's Disease (AD), because they are the precursors of the A-beta peptide found in the amyloid deposition of all AD patients, and because certain forma of familial AD (FAD), are genetically linked to specific AP mutations. Soluble non-amyloidogenic truncated APP (solAPPtrunc), without the cytoplasmic and transmembrane sequence, is produced when full length APP is cleaved by "secretases". However, using antibodies specific for the cytoplasmic APP, we detect in the lumen of bovine chromaffin granules (CGs) a soluble antigen that immunocytochemically and on SDS-PAGE was very similar to the full length APP. A similar APP was also released in vitro from membranes, in a process mediated by an enzymatic mechanism. These soluble proteins are designated here solAPPcyt for soluble APP containing the cytoplasmic sequence. A solAPPcyt of about 130 kDa was detected in the conditioned media of both chinese hamster ovary (CHO), and primary chromaffin cell cultures. Secretion of this APP from primary chromaffin cells was regulated by depolarization and cholinergic agonists. Therefore, full length APP seems to assume a transmembrane and a soluble topology. SolAPPcyt is potentially amyloidogenic and structural considerations suggest that it may actually be the proximate precursor of A-beta. Here, we propose to study the structural differences between membrane bound (transmembrane) APP and solAPPcyt, the specificity and regulation of the cellular processes involved in the production solAPPcyt and its relationship o A-beta, and the APP sequence elements responsible for the secretion of solAPPcyt. Recently, we obtained evidence that APP species carrying FAD-linked mutations, produce significantly higher amounts of solAPPcyt than wild type APP, suggesting that these mutations may interfere with the APP membrane interactions and ultimately cause membrane damage. We propose to use transfected cell lines to study the effect of all FAD-linked mutations on the secretion of solAPPcyt. In addition, we will study whether cell lines form sporadic or FAd patients, secreted higher levels of solAPPcyt than control cell lines, and will determine the levels of solAPPcyt in normal and AD patients. We will use an in vitro system developed in our laboratory to determine the amounts of solAPPcyt released from membranes prepared from control and AD patients and from AD and normal cell lines. The study will elucidate the structure of solAPPcyt and the mechanism of its production and secretion, and will examine its relationship to AD and A-beta.